Screening Of Virulence Factors Related To Blood-brain Barrier Breakthrough By Enterococcus Faecalis

[Objective]Enterococcus faecalis is an ancient microorganism,which is a common flora in the digestive tract of humans and animals.It occasionally colonizes the oral cavity and vagina,and is an opportunistic pathogen.However,as the pathogenicity of Enterococcus faecalis increases year by year,the number of cases caused by Enterococcus faecalis is also increasing year by year,especially the case of meningitis caused by Enterococcus faecalis,which undoubtedly brings serious health problems to humans and animals.threaten.At present,in the studied cases of bacterial meningitis,virulence factors play an important role in causing inflammation and destroying the permeability of the blood-brain barrier.still not clear.In this study,by constructing a genome-wide random display library of E.faecalis causing E.faecalis,we screened out the virulence factors that play a role in the process of E.faecalis breaking through the blood-brain barrier.Lay the foundation.[Methods](1)The blood-brain barrier model was established to observe the effect of different Enterococcus faecalis on the permeability of the blood-brain barrier.(2)Construction of a genome-wide phage random display library of Enterococcus faecalis meningitidis.(3)Screening of relevant virulence factors in the process of breaking through the blood-brain barrier based on the phage display library.[Result](1)The mouse primary microvascular endothelial cells and primary astrocytes were successfully isolated,and the cells were identified by light microscopy and fluorescent staining.Based on the Transwell cell culture model,an in vitro blood-brain barrier model was constructed,and 4 The permeability of the blood-brain barrier model was verified by h penetration test,fluorescein sodium penetration test,and horseradish peroxidase penetration test.The bilayer cell blood-brain barrier model had lower permeability.After selecting different Enterococcus faecalis to interact with the blood-brain barrier model,the XJ6 strain of Enterococcus faecalis could significantly change the barrier permeability.(2)DNA fragmentation was performed under different fragmentation conditions.Under the conditions of 50%power,fragmentation for 2 s,and an interval of 10 s,the DNA could be fragmented into fragments of 300-3000fragments,which were ligated with plasmids and electrotransferred into competent E.coli.Based on phage display technology,M13 phage constructed a random display library of Enterococcus faecalis whole genome phage with a capacity of 2.7×10~4.(3)The in vitro cell screening method in the phage display screening method was selected,and the brain microvascular endothelial cells and astrocytes in the blood-brain barrier model were used as target cells to interact with the mouse in vitro blood-brain barrier model through the phage random display library.Through multiple screening,elution,and enrichment,a total of69 positive phage clones were obtained,and the positive clones were picked for expansion and culture.protein,collagen adhesion protein,surface anchoring protein,gelatinase-like protease,RNA polymerase,surface protein.